Our work continued on the interactions of metabolites with mammalian protozoan cells. Our major projects were the following: 1. Continued work on the mechanism of de novo purine and thymidylate synthesis inhibition by antifolates, including methotrexate, has strengthened a novel postulate that inhibition of metabolic pathways results from direct enzyme inhibition rather than via an indirect mechanism of folate depletion. These studies illustrates that de novo purine and thymidylate synthesis are inhibited by clinically relevant concentration of methotrexate without significant folate depletion in two human cell lines (breast MCF-7 and promyelocytic from normal human volunteers. These studies further suggest that the mechanism of metabolic inhibition is through direct inhibitory effects of accumulated dihydrofolate polyglutamates on the folate-requiring thymidylate synthase and AICAR transformylase. This work has been supported by corroborating the relative folate preservation during antifolate exposures using a highly sensitive and specific assay for intracellular folates. This assay was developed by Dr. Priest and is capable of directly measuring folates without the need for radiolabels with their attendant deficiencies. 2. During the course of the above studies, a novel physiologic folate was discovered, 10-formyl-dihydrofolate. Continued investigations have illustrated the formation of this compound in breast cells and in normal human myeloid progenitor cells. Identification and characterization of the enzyme responsible for the formation of dihydrofolate has been accomplished and the effects of this folate on folate-requiring